This proximal tubule (PT) is the major site of HCO3, reabsorption/acid secretion in the mammalian kidney. Although the classical model of PT acid secretion includes only a Na-H exchanger at the luminal membrane of the PT cell and a pathway for HCO3 exit at the basolateral (i.e., blood-side) membrane, we now know that this is an oversimplification in two respects. First, numerous acid-base transporters have been identified in PT cells. Nevertheless, several of these remain to be characterized. Second, there are important qualitative and quantitative differences in acid-base transport among PT cells, based upon both distance along the nephron from the glomerulus (i.e., S1 vs. S2 vs. S3 segments) and the location of the glomerulus giving rise to the tubule (i.e., superficial, SF vs. juxtamedullary, JM). Understanding PT acid-base physiology will require a systematic characterization of each acid-base transpor-ter in each of the six PT subtypes (SF S1-S3, JM S1-S3), a task that has just begun. We propose to continue our characterization of intracellular pH(pHi) regulation and acid-base transporter in experiments on isolated perfused rabbit PTs. pHi will be continuously monitored by loading the cells with a pH-sensitive dye, illuminating a small number of cells with a 10-mu m- diameter beam of light, and measuring either the dye's absorbance or fluorescence. We will assess individual acid-base transporters by monitoring the recovery of pHi from acute acid or alkali loads, or by monitoring rates of pHi change after other perturbations. One aim is to examine the properties of acid-base transporters that are not well characterized in intact tubules, initially employing only one PT subtype (e.g., SF S3). These transporters include monocarboxylate cotransporters; the Na/HCO3 cotransporter in the S3 segment; transporters or inorganic phosphate, amino acids and other solutes; and the novel alkalizing process activated by basolateral HCO3, in the S3 segment. Our second aim is to conduct a systematic and comprehensive survey of each established PT acid- base transporters in each of the six PT subtypes. Our third aim is to analyze the effects of angiotensin II and other agents not only on luminal Na-H exchange and basolateral Na/HCO3 cotransport, but on the whole ensemble of transporters that affect pHi in the PT. Finally, our fourth aim is to use newly available Na+-sensitive dyes (and possibly voltage- sensitive dyes) to study Na+- (and voltage-) coupled acid-base transport in the rabbit PT. The proposed work will provide a comprehensive description of acid-base transport throughout the PT. The use of isolated, perfused tubules will insure that the expression of the transporters is as normal as possible, and that anatomical origin of the tubules is precisely known.